Association among metabolic status, oxidative stress, milk yield, body condition score and reproductive cyclicity in dairy buffaloes

Abstract The aim of this study was to determine the metabolic status, stress and ovarian activity through progesterone profile in dairy buffalo as influenced by post‐partum days, body condition score (BCS) and lactation number. A total of 45 dairy buffaloes were involved and divided into three factors based on their BCS (2.5, 3.0 and 3.5, respectively), lactation number (1, 2 and 3, respectively), and post‐partum intervals (14, 28, 42 and 56, respectively). Based on findings, from day 14 to day 56 after parturition, blood glucose, cholesterol and triglyceride levels increased significantly (p < .01), while malondialdehyde (MDA) and cortisol levels decreased significantly (p < .05) on day 56 of the trial. With increased BCS levels, milk yield, blood metabolites and progesterone improved significantly (p < .05), whereas stress indicators decreased significantly (p < .01). Blood metabolites, progesterone and milk production were significantly (p < .01) higher and stress indicators (MDA and cortisol) were significantly (p < .01) lower in lactation stage. Buffaloes with a greater lactation stage and growing post‐partum stage had better blood metabolite and progesterone concentrations and less stress. It was concluded that better BCS and increased lactation stage have significant impact on milk yield, stress reduction and restoration of ovarian activity in buffaloes during post‐partum period.


| INTRODUC TI ON
The transition period, which lasts 21 days before and after parturition, is the most essential period for dairy animals. Dairy cows are sensitive to metabolic problems and oxidative stress during the physiological phases of pregnancy and lactation (Ullah et al., 2020).
The peri-parturient phase has a significant impact on the health and subsequent performance of dairy animals since they undergo significant metabolic and physiological changes during this time (Alharthi et al., 2021;Piccione et al., 2012). Negative energy balance (NEB) during early lactation is one of the common physiological causes of oxidative stress and health concerns in dairy animals (Elsayed et al., 2017;Piccione et al., 2012;Saqib et al., 2018;Ullah et al., 2019b).
The BCS is a quick and easy way to analyse an animal's energy reserves by measuring fat metabolism and its relationship to energy metabolism (Hoedemaker et al., 2009;Shah et al., 2019;Ullah et al., 2019a). Since BCS is related to the fertility status of dairy cows (Laubenthal et al., 2017). Cows having a greater BCS at calving exhibited higher fatty acid concentrations in early lactation than cows with a lower BCS (Arfuso et al., 2016). Oxidative stress is more responsive in cows with a higher BCS (Alhidary et al., 2016a(Alhidary et al., ,2016bSaqib et al., 2018). Oxidative stress is caused by an imbalance between reactive oxygen metabolites and antioxidants (Ullah et al., 2019b) and various oxidative status markers such as malondialdehyde (MDA) were found to be greater in cows with higher BCS before calving.
It is well known that blood metabolites and BCS levels in dairy animals substantially change during early lactation. These modifications could reveal crucial details regarding reproduction and herd management (Bernabucci et al., 2005;Kim & Suh, 2003). Pre-partum BCS and its effects on post-partum blood metabolites, oxidative stress, fertility and milk production have gotten very little attention in dairy buffaloes. The hypothesis of the present research was how the metabolic, stress and ovarian activity via progesterone fluctuation are affected by post-partum period, BCS and lactation stage. Therefore, the present study was designed to evaluate the impact of perceiving BCS and lactation numbers on metabolic status, milk yield, stress indicators and resumption of ovarian activity through progesterone profile in dairy buffaloes during the post-partum period.

| Selection of animals
A total of 45 multiparous dairy buffaloes (5-7 years) were involved and divided into three groups based on BCS (2-2.5, 2.5-3.0 and 3.0-3.5), lactation stage (1, 2 and 3), and post-partum days (14,28,42, and 56) 21 days after calving. The buffaloes were fed on cutand-carry fodder system that included seasonal green fodder, wheat straw, and cotton seed cakes, covering their nutrients requirements as recommended by NRC (1978). Water was available ad libitum. The ingredients and nutrients composition are given in Table 1 During the sampling period, the physiological parameters (BCS, general health status and milk yield) of the buffaloes were recorded. The BCS was calculated using the score method outlined by Peters and Ball (1987) briefly described as: score 2.0-2.5 -weak; when round transverse processes were observed and palpated with thin fat covering; score 2.6-3.0 -normal; when firm pressure was needed to feel individual transverse processes due to good muscular and fat covering; score 3.1-3.5 -slightly fat; when transverse vertebral processes could not be felt during palpation with firm pressure.
Health status was evaluated with general characteristics of healthy animal, which behaves normally and stays with herd, bright eyes and moist muzzle without discharge from nose no wounds and eating normally. Visiting veterinary practitioners, fellow farmers, and other services providers helped to build farming techniques based on communicative expertise and some engagement. Buffaloes were clinically examined weekly after parturition for rectal temperature, heart rate and respiration rate, external and internal genitalia (data not shown).

| Blood and milk sampling and analysis
During the diestrous period, blood samples were taken on postpartum days 14, 28, 42 and 56, respectively. Early in the morning before feeding, the blood samples were drawn from the jugular vein of dairy buffalos with a disposable syringe. The blood sample was poured Daily milk yield was measured by emptying cow udder completely through gentle hand milking early in morning while practicing same procedure at evening at day 14, 28, 42 and 56, respectively.
Combining the weight of both times milking were used to calculate the daily milk yield.

| DISCUSS ION
In the available published literature, studies have mostly been published in dairy cows during post-partum period. Little attention has been given to the productive and reproductive performance in dairy buffaloes during post-partum period. The objective of the present study was to understand the impact of pre-partum BCS and lactation numbers on metabolic attributes, milk yield, stress indicators and resumption of ovarian activity in dairy buffaloes during postpartum period. Changes in biochemical indices occur as a result of increased metabolic demands during both pregnancy and lactation, despite haemostatic systems that function to maintain blood parameters within physiological levels (Piccione et al., 2012). The need for the regulatory mechanism, responsible for the process involved in milking increases throughout the peri-partum period. As a result, most animals experience distinct alterations in lipid metabolism during pregnancy and lactation (Roche et al., 2009).

TA B L E 2 Metabolic, stress and reproductive indicators of buffaloes on different post-partum days
In the current study, the levels of blood metabolites increased significantly as the number of post-partum days grew from 14 to 56, resulting in higher blood levels of glucose, cholesterol and triglycerides and lower levels of stress markers (MDA and cortisol) during the phase of uterine involution. It reflects the recovery of the recently parturited buffaloes (resumption of ovarian activity) from parturition stress as mentioned through progesterone profile . The dietary quality, management, BCS, parity, lactation number and climatic conditions affect the post-partum blood metabolites and stress indicators (Ciaramella et al., 2005;Habib et al., 2007;Khan et al., 2016). During the post-partum period, dairy buffaloes go through a lot of physiological and hormonal changes. Negative energy balance is easily developed in the early post-partum stage due to metabolic stress induced by a fall in dry matter intake and increased energy demands at calving and the start of lactation (Mishra et al., 2007). In the current study, glucose concentrations were shown to be greater in animals with a good body condition score.
The concentration of glucose rises dramatically as the number of post-partum days increased. Several researchers have supported the idea that blood glucose concentration indicates an animal's energy level (Dokovic et al., 2012;Kumar et al., 2010). Previous research reports (Hagawane et al., 2009;Mandali et al., 2002) found a direct link between glucose availability, production and reproductive activities in lactating animals throughout the post-partum period. During the post-partum period, animals with a satisfactory body condition score had stable blood metabolites levels, with the mammary gland using around 80% of the circulating blood metabolites for milk synthesis (Borghese and Moioli, 2011).
Serum cholesterol in the current study increased significantly as the number of post-partum days increased. Cholesterol is a precursor to the steroid hormones, and an optimal level of blood cholesterol is essential to regulate physiological activities. Animals having good body condition score showed increased cholesterol level. The current findings were consistent with the previous studies reporting a higher level of blood cholesterol during the post-partum period (Hagawane et al., 2009). In the present study, serum triglyceride level significantly increased with increasing post-partum days. Triglyceride concentrations were high in animals having good body reserves (Piccione et al., 2012). Moreover, triglycerides concentrations represent good physiological conditions of the dairy animals (Zhao et al., 2019). The voluntary feed intake and proper nutrition has been strongly associated with increase in triglycerides level as reported in earlier studies (Bertics et al., 1992;Marai et al., 2004). Higher serum triglycerides concentration is associated with increased milk production (Ashmawy, 2015; Civelek These two factors assist the dairy buffaloes to reach the level of conception in the next phase as indicated from the progesterone level. The stress indicators (MDA and cortisol) decreased when the BCS increased from 2.5 to 3.5 in this study. MDA signifies oxidative stress in animals, while cortisol represents the overall stress (Sampath et al., 2004). The imbalance between reactive oxygen species generation and antioxidant availability is referred to as oxidative stress (Majid et al., 2015;Rahman et al., 2014). This decrease in stress is most likely linked to an increase in blood progesterone levels. In this study, greater cortisol levels were linked to lower progesterone levels and vice versa. As a result, our findings support the alternate functions of TA B L E 5 Metabolic, stress and reproductive indicators of buffaloes as affected by lactation number and post-partum days  Abbreviations: DMY, daily milk yield; MDA, malondialdehyde.